Experiment 4
Acid-Base Extraction: Separation of a
Four-Component Mixture; Thin Layer
Chromatography
Solubility Properties of Organic Compounds
Introduction
An understanding of the principles of solubility is of great practical importance in the organic laboratory.
Since most organic reactions are run in solution, knowledge of solubilities will help in choosing the
appropriate solvent.
For example, water cannot be used as a solvent in performing the
dehydrobromination reaction of 1-bromo-1,2-diphenylethane, shown below, since this starting material
is not soluble in water. However, since both reactants: the organic bromide and KOH, are soluble in
ethanol, the reaction can be done in ethanol as the solvent. An understanding of solubility is also helpful
in isolating the product of a reaction. In this example water can be added after the reaction has taken
place. The product (trans-1,2-diphenylethene) is insoluble in water but ethanol and the byproduct (KBr)
are soluble in water. The ethanol and KBr dissolve in water but the product precipitates and may be
isolated by vacuum filtration.
KOH
solvent
Br
1-bromo-1,2-diphenylethane
trans-1,2-diphenylethene
An understanding of solubility principles is useful in conducting many operations in the organic
laboratory. For example, suppose you are attempting to synthesize 1-naphthoic acid (below). While the
reaction is being done, a solid separates from the reaction. Is this material the product, reactant, or an
inorganic salt? Information that may help to answer the question may be obtained by isolating the
insoluble material by filtration. You add water to this material and it dissolves. Based on the structure
of 1-naphthoic acid, which contains two non-polar rings, you would expect it to be water-insoluble.
Because the material is water-soluble, you have not isolated 1-naphthoic acid but a water-soluble salt.
However, if the isolated material were water-insoluble but soluble in aqueous NaOH, then this is
evidence, though not proof, that you have isolated 1-naphthoic acid since carboxylic acids form water-
68
soluble salts when reacted with base.
CO2H
insoluble in water
soluble in aqueous NaOH
1-naphthoic acid
Principles
Solubility refers to the process by which a solute (solid or liquid in most cases) dissolves in a liquid
giving a homogeneous solution. The solubility of a substance is a matter of degree. The solubility of
some substances is so small that they are considered insoluble for all practical purposes whereas other
materials are infinitely soluble (For example, ethanol in water. Actually, we say ethanol and water are
"miscible" [or mixable] meaning they form a solution at any mole ratio). Materials between these two
extremes are partially soluble. Where is the line drawn between soluble and insoluble? A somewhat
arbitrary convention is that materials that are more soluble than 3 g per 100 mL solvent are considered
soluble and those that are less soluble are considered insoluble. There may be borderline cases and other
conventions may be used.
Water Solubility
The principles of solubility are based on intermolecular forces. These should be reviewed from your
general chemistry and organic chemistry textbooks. London forces are the weak intermolecular forces
of a hydrocarbon, such as p-xylene (below). Hydrogen bonding is a strong attractive force in water.
When p-xylene is mixed with water no meaningful solution is achieved because p-xylene cannot
hydrogen bond with water. The interactions of p-xylene with water
CH3
are not of a sufficient strength to incorporate the molecule into the
water lattice. The entropy of the solution would be substantially
reduced with p-xylene in the lattice, increasing the free energy of
water-insoluble
solution. Thus, p-xylene is effectively "squeezed out", forming a
separate layer. While water can interact with p-xylene through
CH3
London forces, p-xylene remains as an insoluble top layer since it
p-xylene
is less dense than water.
If a compound possesses functional groups that are capable of hydrogen bonding with water, then its
water-solubility will increase. For example, aniline is partially water-soluble since it can hydrogen bond
with water in two ways as shown on the next page. The extent of water-solubility is a good estimate of
the ratio of non-polar hydrocarbon content to the number of polar functional groups. A compound could
have few carbon atoms and be water-insoluble, such as 2-bromobutane, or a larger number of carbon
atoms and be water soluble, such as glucose. The solubility depends on this ratio. Generally,
compounds that have one polar functional group will be soluble up to 5 carbon atoms, beyond that they
are insoluble. Common functional groups that are capable of hydrogen bonding, and thereby increasing
water solubility, are: alcohols, carboxylic acids, amines, aldehydes, ketones, ethers, esters, amides.
H
H N
δ
H
H
.. H
O
.. δ
δ
O
69
H
H
H N
δ
Solubility in Organic Solvents
The principles that determine the water-solubility of an organic compound may also be used to
understand the solubility of an organic compound in organic solvents. For example, a nonpolar solvent
such as toluene would be expected to dissolve a non-polar substance such as decalin since they both
share London forces. Therefore, decalin is quite soluble in toluene.
CH3
toluene
decalin
If the number of polar groups in the organic molecule increases relative to its hydrocarbon content then
the compound's solubility in a nonpolar solvent, such as toluene, will decrease. For example, salicylic
acid is sparingly soluble in toluene because of the polar carboxyl and phenolic groups. Because most
organic compounds have at least one polar functional group the best solubility is achieved with slightly
polar organic solvents. For example, chloroform (CHCl3) is an excellent solvent for many polar
compounds such as caffeine whereas carbon tetrachloride or cyclohexane is not.
CO2H
polar end
OH
O
CH3
N
N
non-polar end
Extraction
N
N
CH3
O
CH3
caffeine
salicylic acid
Extraction, one of the oldest purification techniques, has found widespread use in organic chemistry.
70
It is frequently used to isolate an organic compound from a reaction mixture or from a natural source
such as a plant or animal. When you prepare a cup of tea or coffee you are performing an extraction.
In this experiment the components of a mixture of organic compounds will be separated by
performing a series of extractions.
Principles
Extraction is a separation technique that takes advantage of the difference in solubility a compound
exhibits in two mutually immiscible solvents. Most commonly, one of the solvents is water and is
called the aqueous phase; the other solvent is organic and is therefore called the organic phase. A
compound will distribute itself between the aqueous and organic phases according to the solubility it
has in each phase. The ratio of these solubilities is called the distribution coefficient, KD. It can be
determined by simply looking up the solubilities of the compound in the two solvents. For example,
the solubility of benzoic acid is 0.34 g per 100 mL water and 11.0 g per 100 mL toluene. Therefore:
KD
=
=
solubility in toluene / solubility in water
(11.0 g/100 mL)/(0.34 g/100 mL) = 32
Although these solubility data represent conditions of saturation, even when we are not at saturation,
the solute will distribute itself according to KD, even if the volume of the two solvents are not
identical. As an example, we can calculate how 5.0 g of benzoic acid will distribute itself between
100 mL of water and 50 mL of toluene:
KD =
[(5.0 - x)g/50 mL toluene]/[x g/100 mL water]
x
=
0.29 g benzoic acid in water
5.0-x = 4.7 g benzoic acid in toluene
=
32
This calculation shows that when water comes in contact with a solution of benzoic acid in toluene,
most of the benzoic acid remains in the toluene and a small amount dissolves in water. Water is
therefore ineffective in extracting the benzoic acid. It would require repeated extractions with water to
extract the benzoic acid from a toluene solution. In each extraction a small amount of benzoic acid
would extract into water. Combining each water extract would eventually add up to a complete transfer
of the benzoic acid from toluene to water but the volume of water would be large. Clearly this would be
an inefficient process.
Because many compounds are somewhat soluble in both water and an organic solvent it may be
necessary to repeat the extraction in order to extract the desired amount of material. The relationship
between grams of extracted material and the number of extractions is shown in the following graph.
The calculations for the data points are based on equal volumes of water and organic solvent and 10 g
of an organic compound. Notice that when K =1 it requires four extractions to extract approximately
9.2 g of the material into the organic phase. But when K = 4 only 1.7 extractions will extract the same
amount of material, though extractions can only be done in whole numbers. When K = 4 and four
extractions are done 9.9g out of 10 g has been extracted. One may conclude that with larger KD’s fewer
extractions are needed.
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Grams extracted vs number of extractions
(10 g of organic substance; equal volumes H2O/solvent)
10
l
l
Grams extracted
9
K=4
l
l
l
8
l
K=2
7
K=1
6
5
1
2
3
Number of Extractions
4
In our solubility and recrystallization experiments we learned that the solubility of a compound is a
function of the type of solvent, the structure of the compound, and the temperature. We confirmed the
general rule that a compound is most soluble in a solvent that is most like it in structure and polarity,
i.e. "like dissolves like". Thus, non-polar compounds would dissolve better in solvents such as hexane
and toluene, moderately polar compounds in methylene chloride, chloroform, and diethyl ether, and
highly polar compounds in ethanol. We have a steady progression in these solvents from weak London
forces, to dipolar interactions, to hydrogen bonding. Ionic compounds will be most soluble in water in
which the ions are hydrated.
Choice of an Extraction Solvent
The selection of an organic extraction solvent involves the following criteria:
69
1.
2.
3.
4.
It must readily dissolve the substance to be extracted yet only be sparingly soluble in the other
solvent (usually water).
It should extract little or no impurities or other substances present.
It should not react chemically with the solute in an undesirable way.
It should be easily separated from the extracted solute after extraction. This requirement
usually implies sufficient volatility for removal by distillation or rotary evaporation.
The table below lists common organic extraction solvents and their physical properties. The solvents
must be treated with a degree of caution. For instance, benzene, chloroform, and carbon tetrachloride
are carcinogenic in test animals. Diethyl ether is highly flammable and may develop explosive
peroxides on long standing. Hexane, like diethyl ether, is flammable, only less so. Methylene chloride
is quite volatile (b.p. 41oC), is not flammable, though high levels of prolonged exposure are
carcinogenic in test animals, but to a lesser degree than CHCl3. Whatever organic solvent is used in
the extraction, it should be handled with caution in a well-ventilated hood.
Common Extraction Solvents
Boiling point
o
C
Density
g/mL at 20 oC
Flammable?
C2H5OC2H5
35
0.71
Very
CH2Cl2
41
1.34
No
(CH3)3COCH3
56
0.74
Yes
Chloroform
CHCl3
61
1.49
No
Hexane
CH3(CH2)4CH3
68
0.66
Yes
Ethyl acetate
CH3CO2CH2CH3
77
0.90
Yes
Benzene
C6H6
80
0.88
yes
Solvent
Ethyl ether,
"ether"
Methylene
chloride
Methyl t-butyl
ether
Structure
Extraction as Part of the Work-up
The organic compound to be isolated from a reaction mixture is frequently extracted into an organic
solvent leaving water-soluble inorganic salts behind in the aqueous phase. An illustration of this can be
seen in considering the procedure to isolate cyclohexene in the following dehydrobromination reaction.
The organic product, cyclohexene, is produced along with potassium bromide and water. The work-up
of the reaction, a part of the experimental procedure used to isolate and purify the product,
consists of transferring the reaction to a separatory funnel, adding water to dissolve the potassium
bromide and ethanol, and separating the water insoluble cyclohexene. Distillation of the impure
cyclohexene would further purify the product by removing residual bromocyclohexane and ethanol.
Br
KOH / EtOH
+
70
KBr
+
H2O
Extraction of a Carboxylic Acid
The water-solubility of an organic compound which contains an acidic or basic functional group can be
substantially altered by treatment of the compound with an inorganic base or acid, respectively. For
instance, a carboxylic acid that is insoluble in water, can be made water-soluble by treatment with
aqueous sodium hydroxide, sodium bicarbonate, or sodium carbonate. The reaction of a carboxylic
acid with sodium hydroxide is shown below.
O H
O
C
O
O H
O
C
Na
H2O
NaOH (aq)
1-naphthoic acid
sodium 1-naphthoate
water insoluble
water soluble
By mixing an organic solution of a carboxylic acid with dilute aqueous sodium hydroxide, the
carboxylic acid reacts according the equation above forming the sodium salt which moves into the
aqueous layer because of its water solubility. Thus, carboxylic acids can be separated from other
organic compounds.
Not all salts of carboxylic acids are soluble. If the hydrocarbon portion of the molecule is very large the
hydration of the polar carboxylate group is insufficient to dissolve the salt. The carboxylic acid below, for
most practical purposes, is insoluble in aqueous base even though it forms a salt.
CO2H
CO2 Na
NaOH
H2O
water insoluble
water insoluble
To recover the carboxylic acid the above reaction is reversed by the addition of a strong mineral acid to
neutralize excess NaOH and protonate the carboxylate ion. If the organic acid is water-insoluble, it can
then be isolated by vacuum filtration.
O
H
O
C
H O
H
O
71
C
O H
H2O
Extraction of a Phenol
Phenols (pKa’s near 10) are not as acidic as carboxylic acids (pKa’s near 4-5) and do not form salts with
sodium bicarbonate. However they will react with sodium hydroxide. This difference in acidity can be
used to separate a phenol from a carboxylic acid by extraction with sodium bicarbonate. The
carboxylic acid forms a water-soluble salt and the phenol remains in the organic phase. The phenol can
then be extracted into the aqueous phase with sodium hydroxide. The reaction between a phenol and
sodium hydroxide is shown below. The phenol may be recovered by adding acid to protonate the
phenoxide ion.
O
H
O
H
NaOH (aq)
water-soluble salt
OCH3
vanillin
OCH3
O H
O Na
H2O
O H
Extraction of an Amine
An amine can be extracted in an analogous manner by treatment with aqueous acid. Treatment of the
amine with a strong acid forms the water-soluble salt of the amine as shown below.
H
N H
H O H
H
+
N
H
O H
H
water soluble
water insoluble
The water-soluble amine salt can be isolated from the strongly acidic medium by addition of enough
strong base to neutralize any residual strong acid and deprotonate the amine salt. The free amine can be
isolated by vacuum filtration if it is a solid.
72
H
O H
N H
N
H
water soluble
+
O H
H
water insoluble
Functional Groups that Form Salts Quantitatively
You should note that amines, carboxylic acids, and phenols are the only common functional groups that
are sufficiently basic or acidic to form salts quantitatively in aqueous solution. This results from their
pKa’s and the fact that the strongest acid in water is the hydronium ion and the strongest base in water is
hydroxide ion. You may write acid-base reactions for alcohols, esters, etc. but the salts will not be
formed quantitatively when water is present. Do not confuse acid-base properties with normal
properties of organic compounds. For example, 2-propanol dissolves in dilute NaOH but not because
of acid-base chemistry.
Flow Charts
A flow chart is a pictorial representation of what takes place in an operation. In this case it applies to
an acid-base extraction. The structures of the molecules in the mixture appear at the top of the flow
chart. The first vertical line shows an operation. In the example below this means that the mixture is
dissolved in ether, placed in a separatory funnel and shaken up with aqueous acid. A chemical reaction
takes place at this point. The next horizontal line refers to the two immiscible phases. On the left is the
ether layer with three of the components in solution. On the right is the aqueous layer with one
component dissolved and in the form of a salt. As a general rule the salts will be in the aqueous layer
and the uncharged molecules in the nonpolar (organic) layer. The following flow chart summarizes an
extraction including the compounds, as they exist at each stage of the extraction. Note: The flow chart
below is not identical to what you will be doing in the lab.
You should be able to draw a flow chart for a variety of mixtures in which the separation is based on
acid-base chemistry. You should also be able to write a chemical equation, using arrows, for each acidbase reaction that is a part of the flow chart.
73
H3C
N
CH3
OH
CO2H
Br
dissolve in ether,
extract with acid (HCl)
ether
OH
CO2H
aqueous
H3C
H
N
CH3
Cl
Br
NaHCO3
aqueous
ether
OH
CO2Na
NaOH
H3C
N
CH3
Br
NaOH
ether
HCl
aqueous
O Na
CO2H
Br
evaporate
ether
HCl
OH
if a solid, filter; if
a liquid, extract
with ether and
evaporate.
if a solid, filter; if
a liquid, extract
with ether and
evaporate.
74
if a solid, filter; if
a liquid, extract
with ether and
evaporate.
The Experiment
The extractions and isolations should be done the first week and TLC analysis the second week.
In this experiment the components of a four-component mixture consisting of p-anisic acid, lidocaine,
p-phenylphenol, and biphenyl will be separated by using acid-base extraction. The amine, lidocaine,
will be extracted with aqueous acid, the carboxylic acid, p-anisic acid, will be extracted with sodium
bicarbonate, the phenol, p-phenylphenol, will be extracted with sodium hydroxide. The neutral
compound, biphenyl, will be left in the organic solution.
O
OH
C
H
N
OH
O
OCH3
biphenyl
p-phenylphenol
p-anisic acid
Use of the Separatory Funnel
Proper use of the separatory funnel will be demonstrated in the pre-lab.
The conical shape and narrow neck at the stopcock make separation of
aqueous and organic layers easy. The following directions provide a
general procedure for use of the separatory funnel.
Support the funnel in an iron ring (ideally cushioned with plastic or
rubber tubing). The photograph at the right shows the standard taper
addition funnel being used as a separatory funnel. The diagrams below
illustrate the use of the separatory funnel. Close the stopcock and add to
the separatory funnel the liquids for the extraction using a shortstemmed funnel. Insert the stopper (if glass, it may be very lightly
greased; the yellow cap plug may also be used) and invert the funnel
grasping it with both hands.
75
lidocaine
N
Note that one hand is used to keep the stopper in place! Carefully rock the contents to and fro, once or
twice. Then, with the barrel pointed up into the hood and away from you and your neighbors,
slowly open the stopcock. Doing this will relieve pressure inside the funnel. Pressure buildup is quite
common with volatile solvents such as diethyl ether, methylene chloride, or hexane. Heat generated by
the warmth of the hands or from a reaction in the funnel, such as an acid-base reaction, will volatilize
the solvent. Gas is often generated during extractions, for example, when a carboxylic acid is extracted
with aqueous sodium bicarbonate, as in this experiment, CO2(g) is generated.
After the pressure is released, close the stopcock, shake the funnel
gently two or three times, and again invert the funnel, releasing
the pressure by opening the stopcock. Repeat this process until
pressure buildup is slight. Then shake the contents vigorously for
a short time (10-15 sec.) to thoroughly mix the phases. Always
make sure to hold the funnel in a way to apply pressure to the
stopper, otherwise it may fall out while shaking. Replace the
funnel in the iron ring and remove the stopper. The liquid in the
funnel will not drain properly if the stopper is left in the funnel.
Allow the layers to separate cleanly, then slowly draw off the
lower layer into a flask of appropriate size. As the interface
approaches the stopcock, slow down the flow. Close the stopcock
just as the upper layer enters the stopcock bore. If the upper
layer is to be transferred to another vessel, pour it through the
top of the funnel. Do not run the upper layer through the
stopcock. The relative positions of the aqueous and organic layers
in the funnel will depend on their densities. The more dense
solvent will be the lower layer. The table of extraction solvents
in this experiment can be used to determine which solvent will be
the lower layer. When there is some doubt about which is the organic layer, remove a few drops of the
upper layer and check if it dissolves in water. Note: Before you dispose of any layer in an
extraction, make sure that it is not the one you should keep.
Drying Agents
An organic liquid in contact with an aqueous medium invariably incorporates water. This water must
be removed prior to evaporation of the solvent to insure that what is left is truly dry. A drying agent is
an inorganic solid which, when added to the "wet" organic phase, removes water by incorporating the
water into its own crystal structure. Common drying agents such as magnesium sulfate MgSO4, and
sodium sulfate Na2SO4 can remove up to six and ten waters, respectively, for each formula unit. Thus,
a small amount of drying agent can remove large quantities of water.
Procedures
76
WASTE: This experiment should generate no waste. Aqueous filtrates may be poured down the drain
and the ether solvent will be removed on the rotary evaporator. Isolated components will be submitted
in vials.
Diethyl Ether (Ether) is highly volatile and flammable. Be sure there are no flames in the hood
whenever you work with ether. The acids and bases used in the procedure are corrosive. Always
wash your hands after using them.
The Extractions
(Shake the separatory funnel well in doing these extractions!)
Always use a clean and dry pipet for removing TLC samples.
Assemble the 125 mL separatory funnel on a ring, put the stopcock in the closed position and add 1 mL
of ether to make sure that the funnel does not leak! The stopcock should have a nut, black O-ring, and
washer. If the stopcock fits without leaking, proceed with the experiment. Bring a 50 mL Erlenmeyer
flask to the organic prep-room and you will be given 40 mL of an ether solution containing a mixture of
p-anisic acid, biphenyl, p-phenylphenol, and lidocaine. Using a small conical funnel, pour the ether
solution into the separatory funnel and rinse the flask with several portions (2x 3mL) of ether. Remove
2-3 drop of the solution with a clean pipet and add it to a vial. Secure the lid and label as “mixture”.
Extraction of Lidocaine: Extract the organic phase with a 10 mL portion of 1 M HCl. Drain the lower
aqueous layer into a 30 mL beaker. Place the beaker on a sheet of paper labeled “HCl extract” and set
aside in the hood. Remove 2-3 drop of the ether solution with a clean pipet and add it to a vial. Secure
the lid and label as “after HCl extraction”.
Extraction of p-anisic acid: Extract the organic layer with 10 mL of saturated sodium bicarbonate and
drain into a 50 mL beaker. Repeat with another 5 mL portions of saturated sodium bicarbonate. Place
the beaker on a sheet of paper labeled “bicarbonate extract” and set aside in the hood. Remove 2-3 drop
of the ether solution with a clean pipet and add it to a vial. Secure the lid and label as “after
bicarbonate”.
Extraction of p-phenylphenol: Extract the organic layer with six 5 mL portions of 1 M NaOH (dilute
the 4M NaOH appropriately). This means that the aqueous layer is removed before the next 5 mL
portion is added. The aqueous layers are collected together in a 50 mL Erlenmeyer flask. Place the
flask on a sheet of paper labeled “NaOH extract” and set aside in the hood. Remove 2-3 drop of the
ether solution with a clean pipet and add it to a vial. Secure the lid and label as “after NaOH”.
Isolation of Components (The following may be done in any order.)
Lidocaine: Place the beaker containing “HCl extract” in the ice bath and add 4 M NaOH slowly while
stirring with a glass rod. Add base until the aqueous phase is basic (pH approximately 11 with pH
paper). Cool in the ice bath for 10 minutes with occasional stirring. Do not filter until a definite
crystalline solid is observed. If the solid does not form within several minutes add a seed crystal. When
a solid has formed filter on the Hirsch funnel rinsing with ice water. Remove the solid and filter paper
77
to a piece of paper to dry over the week. When dry remove the filter paper and weigh the solid.
p-Anisic acid: Place the beaker containing “bicarbonate extract” in the ice bath and add 6 M HCl
slowly while stirring with a glass rod. Foaming will occur as CO2 is formed. Add acid slowly until the
aqueous phase is acidic (pH approximately 3 with pH paper). This will require some patience. If the
acid is added too fast the foam will overflow. Cool in the ice bath for 10 minutes with occasional
stirring. Filter on the Buechner funnel rising with ice water. Allow air to pull through the funnel for 2-3
minutes. Remove the solid and filter paper to a piece of paper to dry over the week. When dry remove
the filter paper and weigh the solid.
p-Phenylphenol: Place the flask containing “NaOH extract” in the ice bath and add 6 M HCl slowly
while swirling to mix. Some foaming may also occur. Add acid until the aqueous phase is acidic. Cool
in the ice bath for 10 minutes and filter on the Buechner funnel rinsing with ice water. Remove the solid
and filter paper to a piece of paper to dry over the week. When dry remove the filter paper and weigh
the solid.
Biphenyl: [This may be done the following week] Pour the ether from the separatory funnel into a 50
mL Erlenmeyer flask and dry with 1-2 g sodium sulfate. After 5 minutes with occasional swirling
gravity filter the solution through a cotton-plugged short step funnel into a clean and dry tared 50 mL
round bottom flask . It is important to tare the round bottomed flask because your recovered weight of
biphenyl is based on that. Always use the same balance when taring a flask and weighing it later. Rinse
the flask and sodium sulfate with several small portions of ether (~5 mL) . Evaporate the ether on the
rotary evaporator. Leave the flask open over the week to allow the residual ether to evaporate, then
weigh the flask. Scrape out as much of the biphenyl as possible. Rinse the flask with acetone several
times into the waste container. Then wash the flask using the ultrasonic bath and soap/test tube brush.
Submit the purified four products in labeled vials after the chromatography has been completed.
Thin-Layer Chromatography
Background
Chromatography is an important laboratory method for
separating organic compounds for purification or analysis.
Although originally developed for separating colored
substances of plant origin (chromatography actually means
color-writing), the technique may be applied to the separation
of colorless substances. Chromatography is often the most
effective method for separating compounds which, in many
cases (e.g., small quantities), cannot be separated by the
traditional techniques of distillation and recrystallization. In
fact, chromatography, in a broad sense, is not only important in
organic chemistry but also in many related chemical sciences
such as biochemistry, medicinal chemistry, clinical chemistry,
78